Our recent studies developed acute sequence evolution model which has been applied to interpret sequence clones derived from 102 subjects with acute HIV subtype B infection, from 69 subjects with acute HIV subtype C infection, and from numerous SIV-infected macaques. Our model enabled an assessment of the sequence diversity in HIV infections originating from a single transmitted viral strain and the estimation on the period of infection, which has been a significant and beneficial contribution to HIV research community. We propose to expand our research activity of modeling intrahost HIV diversification toward the development of a novel assay identifying new, recent HIV infections. In the HIV/AIDS prevention field, it is critical to assess how many people have been recently infected in a given area in order to evaluate the feasibility of prevention and intervention trials. The diagnosis of HIV infection is currently possible from blood samples but reliable assays predicting how long an individual has been infected have not been developed. Various detuned antibody assays, or avidity-based assessments, have been used, assuming that antibody titer and avidity increases with time. However, serologic assays are problematic because (i) the rate of maturation of the antibody response varies between different individuals, (ii) some subjects with low CD4 counts or low virus loads may be inaccurately counted as having recent infection, and (iii) serologic assays can be negatively affected if the infecting virus clade differs from the clade of the antigen used in the assay (this is a particular problem in mixed-clade epidemics). This proposal aims to provide empirical and theoretical foundations for inventing a novel assay that distinguishes new, incident infections from chronic, asymptomatic infections. Major innovations of the proposal include (i) its comprehensive integration of next-generation ultradeep pyrosequencing data and biomathematical modeling and (ii) a novel statistical design estimating the number of founder viruses and the duration of infection. Our proposed study will focus on overcoming two primary barriers to the development of a sequencing based assay. First, the assay potentially mis-classifies early infections with multiple distinct founder strains as chronic infections. Second, it is questionable whether the assay can distinguish incident samples from chronic ones obtained at late stages of infections. We propose to collect a large scale of HIV sequence data from diverse population in different epidemic regions and different stages of infections. Ultradeep sequencing data in conjunction with novel statistical designs will lead us to invent a novel assay that is capable of identifying incident infections. The proposed work will advance HIV prevention research by providing a reliable assay based on the characteristics of intrahost HIV diversification.